Alpha arteether resistance domain

ABSTRACT

The present invention relates to an alpha-arteether resistance domain (ADR) of Sequence ID No.1 and a method of identifying ADR in alpha-arteether resistant pathogens and lastly, it relates to set three pairs of primers of sequence ID Nos. 3 to 8.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority on prior U.S. ProvisionalApplication Ser. No. 60/458,376, filed Mar. 31, 2003, which isincorporated herein in its entirety by reference.

REFERENCE TO SEQUENCE LISTING

The present application incorporates by reference a file named: US1374-04 Khanuja Sequence Listing including SEQ ID NO.: 1 to SEQ ID NO.:9, provided herewith in a computer readable form—on a diskette, createdon Nov. 5, 2004 and containing 5,993 bytes. The sequence listinginformation recorded on the diskette is identical to the written (onpaper) sequence listing provided herein.

FIELD OF THE PRESENT INVENTION

The invention relates to a sequence of nucleotides in the DNA ofEscherichia coli gyrase A gene which when changed provides resistance tocc-arteether to the bacteria. This sequence can also be defined as thea-arteether resistance domain and can have wide spread application inthe detection of resistance to a-arteether and related seauiterpeneendoperoxides. This invention also relates to the functional domain ofgyrase A protein and in future can be utilized in structure/target baseddrug designing.

BACKGROUND AND PRIOR ART REFERENCES OF THE INVENTION

Since, ancient times, the plant Artemisia annua (family: Asteraceae) hasbeen used as a traditional Chinese herbal medicine known as Qinghao fortreating fever and malaria. The effective constituent was isolated bythe Chinese investigators in 1972 and shown to be the sesquiterpenelactone, named artemisinin or qinghaosu (Jing-Ming et al., 1979; Tu et<2/.,1981).The structure of artemisinin has now been confirmed by avariety of analytical methods. Because artemisinin posseses highplasmodicidal property, it holds considerable promise for the treatmentof drug resistant malaria. Artemisinin is poorly soluble in water andoil but readily soluble in most aprotic solvents. This property coupledwith its short half-life, as well as desire to improve more potentderivatives, led to the efforts for chemically modifying its structureand synthesizing new artemisinin derivatives. Thus, ether derivatives ofartemisinin called arteethers were prepared from dihydroartemisinin byetherification with ethanol in the presence of Lewis acid.

Absolute stereochemistry of arteethers (a and 6 isomers) at C-12 wasalso determined and it is 2-3 times more potent than artemisinin. Thecompounds a and 8 arteethers were developed as an antimalarial drug inIndia by the Central Institute of Medicinal and Aromatic Plants (CIMAP)and Central Drug Research Institute (CDRI) after phase III clinicaltrials. The arteether is presently sold in the market under the tradename E-MAL (injection).

In our earlier invention, we found a novel selective property of thecompound a-arteether, which is inhibitory against the gyr mutant strainsof E.coli, but ineffective against wild type strains (U.S. Pat. No.6,127,405). Further we also developed in a separate U.S. Pat. No.6,423,741 a strategic and novel combinations of a arteether which can beused as advanced generation drug(s) to counter the resistancedevelopment itself while, having a potential to be used in treatinginfectious diseases particularly in those cases where drug resistantstrains are known to appear very frequently.

The uniqueness and most useful feature is that, in a combination of aarteether and quinolone drugs and likewise, the spontaneous mutantsarising resistant to quinolones or the derivatives will be killed by aarteether and at the same time any a arteether resistant strains becomehighly sensitive to nalidixic acid and hence eliminated by it throughthe combination approach. The new composition of compounds inhibits theresistance development due to mutation in the gyr A gene of bacteria, inwhich one component is a arteether and the other may be nalidixic acidor any of the fluoroquinolones (comprising of Ciprofloxacin,Norfloxacin, Levofloxacin, Sparfloxacin, Oxfloxacin and Lomefloxacinetc.) or compounds of similar nature against which the resistance maydevelop through a related process.

These above inventions were based on in vivo assays and do not indicateany insight into the genome for a arteether resistance mechanism basedon which similar molecules can be structured/designed to target the DNAgyrase enzyme. Person skilled in the art may obtain clues from thepresent invention to design such molecules for a wide spectrum ofmicrobes and hence the commercial importance of the invention.

OBJECTS OF THE PRESENT INVENTION

The main object of the present invention is to identify analpha-arteether resistance domain.

Another main object of the present invention is to develop a method ofidentifying an alpha-arteether resistance domain in a pathogen.

SUMMARY OF THE PRESENT INVENTION

The invention relates to a sequence of nucleotides in the DNA ofEscherichia coli gyrase A gene which when changed provides resistance tocc-arteether to the bacteria. This sequence can also be defined as thea-arteether resistance domain and can have wide spread application inthe detection of resistance to a-arteether and related seauiterpeneendoperoxides. This invention also relates to the functional domain ofgyrase A protein and in future can be utilized in structure/target baseddrug designing.

DETAILED DESCRIPTION

Accordingly, the present invention relates to identifying analpha-arteether resistance domain, and also, to developing a method ofidentifying an alpha-arteether resistance domain in a pathogen andthereby help in developing drugs against the pathogens.

In main embodiment of the present invention, wherein an oligonucleotideas alpha-arteether resistance domain (ADR) GGTCACTCGGCGGTCTATGAC of SEQID No. 1.

In still another embodiment of the present invention, wherein the domainis from 241 to 261 nucleotide position of gyr A gene from translationstart site of E. Coli.

In still another embodiment of the present invention, wherein anoligopeptide Gly Asp Ser Ala Val Tyr Asp of SEQ ID No. 2, correspondingto an oligonucleotide as alpha-arteether resistance domain (ADR) of SEQID No. 1.

In still another embodiment of the present invention, wherein theoligopeptide is from amino acid position 81 to 87 in gyrase A peptide ofthe enzyme.

In still another embodiment of the present invention, wherein a methodof identifying alpha-arteether resistance domain (ADR) in aalpha-arteether resistant pathogens, to help develop drugs against thepathogen, said method comprising steps of:

-   -   developing alpha-arteether resistant mutant from arteether        sensitive strain,    -   identifying both phenotypic and genotypic characteristics of the        developed alpha-arteether resistant mutant, and    -   identifying alpha-arteether resistance domain (ADR) in an        alpha-arteether resistant pathogens.

In still another embodiment of the present invention, wherein analpha-arteether resistance domain (ADR) is an oligonucleotide of SEQ IDNo. 1

In still another embodiment of the present invention, wherein the ADR isfrom 241 to 261 nucleotide position of gyr A gene from translation startsite of E. Coli.

In still another embodiment of the present invention, wherein the ADRhas corresponding an oligopeptide of SEQ ID No. 2.

In still another embodiment of the present invention, wherein theoligopeptide is from amino acid position 81 to 87 in gyrase A peptide ofthe enzyme.

In still another embodiment of the present invention, wherein a set ofthree pairs of primers of SEQ ID Nos. 3, 4; 5, 6; and 7, 8.

In still another embodiment of the present invention, wherein theprimers of SEQ ID Nos 3, 5, and 7 are forward primers.

In still another embodiment of the present invention, wherein theprimers of SEQ ID Nos 4, 6, and 8 are reverse primers.

The next millenium will visualize a serious problem of antibioticresistance for the antibiotics developed during the previous century andthis is classified as a serious threat in the WHO report on infectiousdiseases. Currently antibiotics provide the main basis of causativetherapy of bacterial infections. However high genetic variability ofbacteria enable them to rapidly evade the action of antibiotics bydeveloping resistance. Thus there has been a continuous search for newand potent antibiotics. The group of fluoroquinolones thus arevulnerable to this phenomena and our emphasis is to search newantibiotic from plant sources. In this context we have invented thesemisynthetic compound ct-arteether and its effect on fluoroquinoloneresistant bacteria (U.S. Pat. No. 6,127,405). The patent (U.S. Pat. No.6,127,405) deals with the resistance developed against quinolone drugsand use of a-arteether to control the infections, but does not speakabout the resistance development against a-arteether itself. Theinventors studied this aspect of resistance development andexperimentally proved that this cross-resistance (quinolone vsarteether) development can be taken care of using the Combinationtherapy, which is not invented in the earlier invention (U.S. Pat. No.6,423,741). As the invention of a-arteether as a agent to kill thequinolone resistant bacteria is new, no one knows about the resistancedeveloping from a-arteether. This led us to the invention of the drugresistance prevention system using a-arteether and a fluoroquinolonedrug. We studied at the DNA level to determine the mutations developedin the gyrase A gene of bacteria Escherichia coli (Kumar, S. 1976.Journal of Bacteriology, 125: 545-555.). This we assumed as the bacteriadeveloped resistance against fluoroquinolones are sensitive toa-arteether and the bacteria resistant to a-arteether are resistant tofluoroquinolones. The fluoroquinolone drugs induce mutation in thegyrase A gene of bacteria and thereby the resistance developed.

There are consistent attempts to determine the mechanism of action ofthe antibiotics and based on which the newer antibiotics are designed bychemical modification of the prototype compounds. Simultaneously,attempts are being made to follow genomic approaches using the genomicdatabase and identifying lethal targets. So, new drugs offluoroquinolones group were developed to target bacterial type IItopoisomerases which are otherwise known as DNA gyrases. Topoisomerasesplay an essential role for the control of the three-dimentional DNAstructure in all cells. Among all topoisomerases bacterial type IItopoisomerase (DNA gyrase) is unique by its ability to introduce thenegative supercoils into covalently closed circular double stranded DNAin the relaxed state. DNA gyrase enzyme has two sub-units A and B. Thequinolones and fluoroquinolones are targeted to the A subunit of theenzyme and shows bactericidal activity against dividing cells. This isdue to the inhibition of the replicative DNA synthesis rather than theprotein or RNA synthesis.

In our experiments planned to detect the biological activity ofarteethers against various strains of E.coli, we found an interestingfeature that among the two isomers, only a isomer of arteether is ableto inhibit the growth of a particular E.coli strain DH5a availablecommercially, which carries a well defined mutation (gyrA 96) in thegene encoding DNA gyrase-A enzyme subunit. As a result of this mutation,the said strain is also resistant to a drug called nalidixic acid. Theother E.coli strains which do not carry gyr mutation were invariablyresistant to a-arteether.

To ascertain the involvement of gyrA mutation, another E.coli strain NK5819 which is also GyrA″ and nalidixic acid resistant was tested forarteether sensitivity. As expected, strain NK5819 was also sensitiveonly to a-isomer of arteether. For further substantiating the Gyr″ anda-arteether resistant relationship in E.coli, yet another strain MTCC482 defective in DNA gyrase B subunit and hence termed GyrB″ was also^tested for arteether sensitivity. Interestingly, even the gyrB MTCC 482strain was susceptible only to a-isomer of arteether. The assays forarteether sensitivity was performed by the standard single diskdiffusion method (Bauer et al, 1966). As a next step, we isolated gyrAmutants of E.coli strain CA 8000, which is otherwise Gyr+ and nalidixicacid sensitive. The gyrA mutants of CA8000 were isolated as nalidixicacid resistant colonies (20 ug/ml), after mutagenic treatment with NTG(100 Hg/ml). The isolated CA8000 gyrA mutants were also sensitive onlyto a isomer of arteether. The above experiments clearly demonstrated thestereospecific inhibition of gyr mutants of E.coli by a-arteether. Onthe other hand, none of the above described gyr mutants were sensitiveto the fl-isomer. To define precisely the involvement of gyr genes onlyin the a-arteether sensitivity, we utilised two recombinant clones (pMK90 and pMK47 containing functional gyr A and gyrB genes respectively) intrans-complementation assays. For this purpose, we mobilized the plasmidclones into the E.coli gyr mutant strains. The resulting transformantswere now nalidixic acid sensitive and a-arteether resistant. Thisconstituted the direct evidence supporting our observation that DNAgyrase enzyme alone is involved in conferring a-arteether sensitivity toE.coli strain. Hence, the gyr strains of E.coli can be used as abiological sensor for detecting the a-isomer of arteether. DNA gyraseenzyme is essential for the bacterial growth. This enzyme transientlybreaks the DNA strands and introduces negative superhelical turns in anATP-dependent process. The E.coli DNA gyrase enzyme is a tetramer withtwo subunits A and B. These two subunits are nalidixic acid andcoumermycin sensitive respectively (U.S. Pat. No. 6,127,405 and aco-pending patent).

The genes (gyrA and gyrB } encoding both these subunits have beenisolated and cloned in E. coli and the prior arts define the drugresistance domain in gyr A gene for quinolones and fluoroquinolones. Butthe prior arts don't describe the resistance domains for a-arteether.

Quinolone Resistance Determining Region

Sequence analysis to DNA from many Bacterial species shows thatresistance mutation tend to alter amino acid near the putative activesite in the gyr A protein (Tyr 122 in E.coli). This region extendingbetween amino acid 67 to 106 is called the QRDR within gyr A of E.coli.Mutations of two codons Serine 83 to a hydrophobic amino acid generallyconfers more resistance than does mutation at position 87.

So in planned experiments we screened and isolated several gyr Aspontaneous mutants resistant to a-arteether from the a-arteethersensitive strains. These a-arteether sensitive strains were selectedrandomly from the quinolone and fluoroquinolone resistant strains. Fromthe genebank database the gyr A gene sequence for Escherichia coli wasdownloaded and forward and reverse primers were designed and synthesizedin ABI 392 DNA synthesizer.

The gyrase A gene of Escherichia coli and the primer sites.

TGGCAAGACA AACGAGTATA TCAGGCATTG GATGTGAATA AAGCGTATAG   −77 GTTTACCTCAAACTGCGCGG CTGTGTTATA ATTTGCGACC TTTGAATCCG   −27 Forward-1                  5′ A ATTTGCGACC TTTGAATCCG 3′                           +1 GGATACAGTA GAGGGATAGC GGTTAGATGAGCGACCTTGC GAGAGAAATT   +24 ACACCGGTCA ACATTGAGGA AGAGCTGAAG AGCTCCTATCTGGATTATGC   +74 GATGTCGGTC ATTGTTGGCC GTGCGCTGCC AGATGTCCGA GATGGCCTGA +124 AGCCGGTACA CCGTCGCGTA CTTTACGCCA TGAACGTACT AGGCAATGAC  +174TGGAACAAAG CCTATAAAAA ATCTGCCCGT GTCGTTGGTG ACGTAATCGG  +224 TAAATACCATCCCCATGGTG ACTCGGCGGT CTATGACACG ATTGTCCGCA  +274 TGGCGCAGCC ATTCTCGCTGCGTTATATGC TGGTAGACGG TCAGGGTAAC  +324 TTCGGTTCTA TCGACGGCGA CTCTGCGGCGGCAATGCGTT ATACGGAAAT  +374 CCGTCTGGCG AAAATTGCCC ATGAACTGAT GGCCGATCTCGAAAAAGAGA  +424 CGGTCGATTT CGTTGATAAC TATGACGGCA CGGAAAAAAT TCCGGACGTC +474 ATGCCAACCA AAATTCCTAA CCTGCTGGTG AACGGTTCTT CCGGTATCGC  +524CGTAGGTATG GCAACCAACA TCCCGCCGCA CAACCTGACG GAAGTCATCA  +574 ACGGTTGTCTGGCGTATATT GATGATGAAG ACATCAGCAT TGAAGGGCTG  +624 ATGGAACACA TCCCGGGGCCGGACTTCCCG ACGGCGGCAA TCATTAACGG  +674 TCGTCGCGGT ATTGAAGAAG CTTACCGTACCGGTCGCGGC AAGGTGTATA  +724 TCCGCGCTCG CGCAGAAGTG GAAGTTGACG CCAAAACCGGTCGTGAAACC  +774 ATTATCGTCC ACGAAATTCC GTATCAGGTA AACAAAGCGC GCCTGATCGA +824 GAAGATTGCG GAACTGGTAA AAGAAAAACG CGTGGAAGGC ATCAGCGCGC  +874TGCGTGACGA GTCTGACAAA GACGGTATGC GCATCGTGAT TGAAGTGAAA  +924 CGCGATGCGGTCGGTGAAGT TGTGCTCAAC AACCTCTACT CCCAGACCCA  +974 Forward-2   5′ATGCGGTCGGTGAAGT TGTGCT3′′3C TTGGAGATGA GGGTCTGGGT GTTGCAGGTT TCTTTCGGTATCAACATGGT GGCATTGCAC CATGGTCAGC +1024 C′5 Reverse-1 CGAAGATCATGAACCTGAAA GACATCATCG CGGCGTTTGT TCGTCACCGC +1074 CGTGAAGTGG TGACCCGTCGTACTATTTTC GAACTGCGTA AAGCTCGCGA +1124 TCGTGCTCAT ATCCTTGAAG CATTAGCCGTGGCGCTGGCG AACATCGACC +1174 CGATCATCGA ACTGATCCGT CATGCGCCGA CGCCTGCAGAAGCGAAAACT +1224 GCGCTGGTTG CTAATCCGTG GCAGCTGGGC AACGTTGCCG CGATGCTCGA+1274 ACGTGCTGGC GACGATGCTG CGCGTCCGGA ATGGCTGGAG CCAGAGTTCG +1324GCGTGCGTGA TGGTCTGTAC TACCTGACCG AACAGCAAGC TCAGGCGATT +1374 CTGGATCTGCCTTTGCAGAA ACTGACCGGT CTTGAGCACG AAAAACTGCT +1424 CGACGAATAC AAAGAGCTGCTGGATCAGAT CGCGGAACTG TTGCGTATTC +1474 TTGGTAGCGC CGATCGTCTG ATGGAAGTGATCCGTGAAGA GCTGGAGCTG +1524 GTTCGTGAAC AGTTCGGTGA CAAACGTCGT ACTGAAATCACCGCCAACAG +1574 CGCAGACATC AACCTGGAAG ATCTGATCAC CCAGGAAGAT GTGGTCGTGA+1624 CGCTCTCTCA CCAGGGCTAC GTTAAGTATC AGCCGCTTTC TGAATACGAA +1674GCGCAGCGTC GTGGCGGGAA AGGTAAATCT GCCGCACGTA TTAAAGAAGA +1724 AGACTTTATCGACCGACTGC TGGTGGCGAA CACTCACGAC CATATTCTGT +1774 GCTTCTCCAG CCGTGGTCGCGTCTATTCGA TGAAAGTTTA TCAGTTGCCG +1824 GAAGCCACTC GTGGCGCGCG CGGTCGTCCGATCGTCAACC TGCTGCCGCT +1874                                  Forward-3  5′TGCCGCT GGAGCAGGACGAACGTATCA CTGCGATCCT GCCAGTGACC GAGTTTGAAG +1924 GGAGCAGGACGAA3′         ′3TAGGA CGGTCACTGG CTCAAAC′5 Reverse-2 AAGGCGTGAAAGTCTTCATG GCGACCGCTA ACGGTACCGT GAAGAAAACT +1974 GTCCTCACCG AGTTCAACCGTCTGCGTACC GCCGGTAAAG TGGCGATCAA +2024 ACTGGTTGAC GGCGATGAGC TGATCGGCGTTGACCTGACC AGCGGCGAAG +2074 ACGAAGTAAT GCTGTTCTCC GCTGAAGGTA AAGTGGTGCGCTTTAAAGAG +2124 TCTTCTGTCC GTGCGATGGG CTGCAACACC ACCGGTGTTC GCGGTATTCG+2174 CTTAGGTGAA GGCGATAAAG TCGTCTCTCT GATCGTGCCT CGTGGCGATG +2224GCGCAATCCT CACCGCAACG CAAAACGGTT ACGGTAAACG TACCGCAGTG +2274 GCGGAATACCCAACCAAGTC GCGTGCGACG AAAGGGGTTA TCTCCATCAA +2324 GGTTACCGAA CGTAACGGTTTAGTTGTTGG CGCGGTACAG GTAGATGACT +2374 GCGACCAGAT CATGATGATC ACCGATGCCGGTACGCTGGT ACGTACTCGC +2424 GTTTCGGAAA TCAGCATCGT GGGCCGTAAC ACCCAGGGCGTGATCCTCAT +2474 CCGTACTGCG GAAGATGAAA ACGTAGTGGG TCTGCAACGT GTTGCTGAAC+2524 CGGTTGACGA GGAAGATCTG GATACCATCG ACGGCAGTGC CGCGGAAGGG +2574GACGATGAAA TCGCTCCGGA AGTGGACGTT GACGACGAGC CAGAAGAAGA +2624 ATAATTTTACTTCTTCATGC CAAAAGGGAG CTATCTCCCT TGTTTGAATT +2674                                 ′3TAGAGGGA ACAAACTTAA GAAAAGTCCAGGCTGCAAAG TCTGGGCTTT TGTCGTATTA GGGCACGGTA +2724 CT′5 Reverse-3AAGTTTGGCT GTGCCCGTAA AAAATGGCTG GCTATACACA AGGAATGTGG +2774 CAATGAGTGGTGAAAAAAAG GCGAAAGGCT GGCGGTTCTA TGGTCTTGTA +2824 GGTTTTGGCG CAATAGCACTGCTTTCCGCT GGCGTCTGGG CGTTGCAATA +2874 TGCTGGCAGT GGGCCAGAAA AAACGTTGTCGCCGCTGGTG GTGCACAACA +2924 ATCTGCAAAT CGATCT +2940Size of the gyr A Gene 2628 Bases

Using the primer pair as mentioned below of SEQ ID No. 3 and 4respectively, 1023 base pair sequence was amplified and sequenced (−48to +975).

Forward-1 5′A ATTTGCGACC TTTGAATCCG 3′ (21 bases) Reverse-15′CTGGGTCTGGGAGTAGAGGTTG 3′ (22 bases)

Using the primer pair SEQ ID No. 5 and 6 respectively as mentioned below993 base pair sequence was amplified and sequenced (−929 to +1921).

Forward-2 5′ATGCGGTCGGTGAAGTTGTGCT 3′ (22 bases) Reverse-25′CAAACTCGGTCACTGGCAGGAT 3′ (22 bases)

Using the primer pair SEQ ID No. 7 and 8 respectively as mentioned below809 base pair sequence was amplified and sequenced (+1868 to 2676).

Forward-2 5′TGCCGCTGGAGCAGGACGAA 3′ (20 bases) Reverse-25′TCAATTCAAACAAGGGAGAT 3′ (20 bases)

Using these primers the total gyrase A gene was amplified to threefragments separately, purified and sequenced in AB1 prism 377 automaticDNA sequencer. The sequences of the mutants were compared to the wildtype.

Development of α-arteether Resistant Mutant from α-arteether SensitiveMutants:

In order to develop an arteether resistant mutants from an arteethersensitive strains a reverse strategy was applied.Quinoline/fluoroquinolone (Q/FQs) resistant strains were developedspontaneously i.e. screening overnight log phase culture of theQuinolone/fluoroquinolone sensitive i.e. arteether resistant strain (Ecoli CA 8000) on the poison agar plate of the Q/FQs. The Q/FQ resistantmutants which appeared on the poison agar plates were first purifiedthrough single colony purification and then checked for their arteethersensitivity. These arteether sensitive mutants on the contrary whenchecked for their Q/Fqs resistance they show different level of Q/FQsresistance.

Bioactivity of Beta-arteether on Selected Q/FQr Mutants Compared to E.coli CA 8000:

The strains of E coli mutants were grown on Luria agar medium andbeta-arteether (250 μg/disc) was applied on disc. The clear zone aroundthe disc killing the bacteria is called Zone of Inhibition (ZOI). Thisis the measure of sensitivity of the strain towards the compoundalpha-arteether.

TABLE 1 Net ZOI in mm Strains of E. coli/Mutants (250 μg/disc) CA8000(gyrA+) — DH5 αgyrA−) 5 ET8000 (gyrA−) 5 NK5819 (gyrA−) 7 CA8001 10CA8002 5 CA8003 9 CA8006 11 CA8007 8 CA8009 14 CA8010 10 CA8012 8Level of Cross Resistance of Different Mutants Compared to E. coli CA8000:

The minimum inhibitory concentrations (MIC) of the quinolone andfluoroquinolone antibiotics were quantified for different strains andcompared among each other. The differential response indicate themeasure of resistance and sensitivity. Higher MIC indicate moreresistance.

TABLE 2 Mutant Nalidixic acid Norfloxacin Sparfloxacin CiprofloxacinLomefloxacin Strains μg/ml μg/mL μg/ml μg/ml μg/ml CA8000 2.0 <0.05<0.05 <0.05 <0.05 DH5a 50 20 10 0.1 0.5 ET8000 100 50 20 0.5 >2.0 NK581920 25 10 <0.1 <0.5 CA8001 150 15 20 <0.1 1.5 CA8002 150 15 20 <0.1 1.5CA8003 150 15 20 <0.1 2.5 CA8005 150 10 20 0.1 0.5 CA8006 150 20 40 0.11.5 CA8007 100 15 20 0.1 1.5 CA8009 100 15 40 0.1 1.5 CA80010 100 15 400.1 1.5 CA80012 100 15 40 0.1 1.5Phenotypic as well as Genotypic Characteristics of the Mutants:

The strains of E coli of the investigation, their phenotypes in terms ofa-arteether (resistance or sensitivity, superscript R or S) orantibiotic resistance or sensitivity, change in nucleotide position ofthe gyrase A gene leading to the phenotype, corresponding amino acidchange in the protein and the position of amino acid change are beingdescribed. Always the phenotype a-arteether resistance is accompaniedwith quinolone or fluoroquinolone sensitivity and vice versa.

TABLE 3 Position of E. coli Change in nucleotide Change in changed S. NoStrains Phenotype (Position) Aminoacid Aminoacid 1 DH5αa-arteether^(S)/Nal^(R) GAC-----AAC(259) Asp-----Asn. 87 2 ET8000a-arteether^(S)/Nal^(R) GAC-----AAC(259) Asp-----Asn. 87 3 NK5819a-arteether^(S)/Nal^(R) GAC-----TAC(259) Asp-----Gly 87 4 CA8001a-arteether^(S)/Nal^(R) Mutant GGT-----TGT(241) Gly-----Cys 81 5 CA8002a-arteether^(S)/Nal^(R) Mutant GGT-----TGT(241) Gly-----Cys 81 6 CA8003a-arteether^(S)/Nal^(R) Mutant GAC-----GGC(260) Asp-----Gly 87 8 CA8005a-arteether^(S)/Nal^(R) Mutant GAC-----GGC(260) Asp-----Gly 87 9 CA8006a-arteether^(S)/Nal^(R) Mutant TCG-----TTG(248) Ser-----Leu 83 10 CA8007a-arteether^(S)/Nal^(R) Mutant TCG-----TTG(248) Ser-----Leu 83 12 CA8009a-arteether^(S)/Nal^(R) Mutant TCG-----TTG(248) Ser-----Leu 83 13 CA8010a-arteether^(S)/Nal^(R) Mutant TCG-----TTG(248) Ser-----Leu 83 15 CA8012a-arteether^(S)/Lome^(R) Mutant GGT-----GAT(242) Gly-----Asp. 81Phenotypic as well as Genotypic Characters (gyr A Gene, QRDR Region) ofthe WT as well as Revertants:

The commercially available strain E. coli DH5□ (Stratagene, USA) issensitive to □-arteether but resistant to quinolone/fluoroquinolone dueto a mutation at 87^(th) aminoacid position of the gyr A protein(Aspartic acid of wild type is changed to Aspargine) with correspondingchange in the codon GAC to AAC. This strain was used to screen□-arteether resistant mutations. The observation was startling as the□-arteether resistant mutants are the exact reversion at the 87^(th)position of the aminoacid of gyrase A subunit i.e. Aspargine changed toaspartic acid, with corresponding change in the codon AAC to GAC.Similarly When the mutants (a-arteether^(S)/Lom^(R)) were searched fora-arteether sensitivity those were found to be exact revertants ataminoacid position 81.

TABLE 4 Position of E. coli Change in Change in changed S. No MutantStrains Phenotype nucleotide Aminoacid Aminoacid 2 AR 9 CA8014a-arteether^(R)/Nal^(S) Mutant AAC-----GAC(259) Asn.-----Asp 87 3 Rev. 1a-arteether^(R)/Lom^(S) Mutant GAT-----GGT(242) Asp-----Gly 81 4 Rev. 2a-arteether^(R)/Lom^(S) Mutant GAT-----GGT(242) Asp-----Gly 81 5 Rev. 3a-arteether^(R)/Lom^(S) Mutant GAT-----GGT(242) Asp-----Gly 81 6 Rev 4a-arteether^(R)/Lom^(S) Mutant GAT-----GGT(242) Asp-----Gly 81 7 Rev 5a-arteether^(R)/Lom^(S) Mutant GAT-----GGT(242) Asp-----Gly 81 8 Rev 6a-arteether^(R)/Lom^(S) Mutant GAT-----GGT(242) Asp-----Gly 81

The α-arteether resistance domain(ARD) can be defined as the domain from241 nucleotide position to 261 nucleotide position of gyr A gene fromthe translation start site (ATG codon) of Escherichia coli correspondingto 81 to 87 amino acid position in the gyrase A peptide of the enzymefrom the N-terminal end. The N-terminal end can be defined as thestarting amino acid in the peptide.

The ARD may extend further beyond the above mentioned nucleotidepositions on either direction of the range mentioned and not limited toEscherichia coli only as the gyr. A gene is highly conserved amongdifferent bacterial species.

The commercially available strain E.coli DH5α (Stratagene, USA) issensitive to α-arteether but resistant to quinoline/fluoroquinolone dueto a mutation at 87^(th) aminoacid position of the gyr A protein(Aspartic acid of wild type is changed to Aspargine) with correspondingchange in the codon GAC to AAC. This strain was used to screenα-arteether resistant mutations. The observation was starting as theα-arteether resistant mutants are the exact reversion at the 87^(th)position of the amino acid of gyrase A subunit i.e. Aspargine changed toAspartic acid, with corresponding change in the codon AAC to GAC.

1. An isolated oligonucleotide consisting of alpha-arteether resistanceconferring domain (ADR) of SEQ ID No. 1.